The present invention relates to a strain of Lactobacillus paracasei subsp. paracasei, a composition thereof for use in food as well as a product containing said strain.
Definition and Characterisation of the Strain
The novel strain (which in the following for simplicity will be designated LMG-P-17806) is a variant of the species Lactobacillus paracasei subsp. paracasei. It has the characteristics of the species with a GC-content of 44%. LMG P-17806 has been isolated from samples from the gastrointestinal micro-flora of humans. LMG-P-17806 is a Gram-positive, homofermentative rod-shaped bacteria. It produces L-lactic acid (laevorotatory stereoisomer of lactic acid) and grows optimally at 37xc2x0 C. The stain is characterized by being tolerant in-vitro against hydrochloric acid and gastric juice by tolerating bile salts without deconjugating them and by having a great ability of assimilating cholesterol. The stain is also characterized by containing three plasmids having a size of 2.2, 4.36 and 9.1 Kb, respectively. Other characteristics are that the strain is fermenting ribose, adonitol, galactose, glucose, fructose, mannose, sorbose, mannitol, sorbitol, N-acetyl-glucosamine, esculin, cellobiose, maltose, lactose, sucrose, trehalose, inulin, melezitose, D-turanose and D-tagatose. On the other hand it does not ferment glycerol, erythritol, D- and L-arabinose, D- and L-xylose, xcex2-methyl-D-xyloside, rhamnose, dulcitol, inositol, xcex1-methyl-D-mannoside, xcex1-methyl-D-glucoside, amygdalin, arbutin, salicin, melibiose, raffinose, starch, glycogen, xylitol, gentiobiose, D-lyxose D- and L-fucose, D- and L-arabitol and 2- and 5-ketogluconate.
The strain has been characterized by SDS gel electrophoresis, in which it has been compared to six other strains of Lactobacillus paracasei subsp. paracasei, vide the accompanying figure. In this comparison it has been shown to differ from all other described strains and at the same time as it when being compared to other lactobacilli appears to belong to the designated species. It has also been characterized with regard to ribosomic RNA in a so called Riboprinter(copyright). With this method the strain has been shown to possess 76% similarity with the type strain for Lactobacillus paracasei subsp. paracasei and 72% similarity to the type strain of Lactobacillus casei analyzed at the same occasion.
The strain has been deposited at Belgian Coordinated Collections of Microorganismsxe2x80x94BCCM, LMG collection, and there been given the accession No. LMG P-17806.
The Advantages of the Strain
LMG P-17806 has, when compared to known strains of Lactobacillus, crucial advantages in the use as probiotics in food and naturopathic medicines, i.e., medical food, by a unique combination of good properties;
the strain has good resistance against gastric juice and bile salts, but unlike many other strains it does not deconjugate the bile salts;
it has a great ability to assimilate cholesterol;
the strain is well managing the passage through the stomach;
the strain has an influence on the conditions in the model of large intestine by increasing the production of L-lactic acid therein;
the strain is not more pro-inflammatory than common yogurt bacteria;
the strain prevents intestinal cells from being invaded by pathogenic microorganisms, such as Salmonella typhmurium;
the strain has an antagonistic action against the gastric ulcer bacterium Helicobacter pylori:
the strain forms bacteriocins which are active against clostridiae;
the strain survives well in milk as well as in frozen and dried form;
the strain, unlike most other lactobacilli has a favourable influence on the taste of fermented milk products (does not give any tang).
The present strain of Lactobacillus paracasei subsp. paracasei can be used as an additive to food or as naturopathic medicine, so called xe2x80x9cMedical Foodxe2x80x9d, or as an additive to naturopathic medicine.
Such medicines can be used for children with the purpose of alleviating atopic problems; for elderly persons in order to correct altered microflora caused by normal alterations by age or an altered secretion of hydrochloric acid; and for persons in general in order to normalize the intestine flora, whereas the content of clostridium bacteria is decreasing, lactobacilli and bufido bacteria being increased and high contents of coliformic bacteria being decreased.
By means of these properties the strain LMG P-17806 differs from previously known strains, which will be shown in the examples below.
Preparation of the Strain
The strain is prepared in the usual way for lactobacilli. A substrate suited for lactobacilli is used. This substrate should for instance contain at least one of the carbohydrates which the strain can ferment according to what is stated above, in combination with proteins, vitamins, minerals and other nutrients which normally are required by lactobacilli. Examples of suitable commercial substrates are yeast extract-glucose broth, MRS (de Man-Rogosa-Sharp broth), Rogosa, milk added with a minor amount of a yeast extract, etc. The strain is cultivated microaerophilicly or in the complete absence of oxygen, suitably at a temperature between +15xc2x0 C. and +42xc2x0 C. If the substrate is grafted with 0.1 to 1% of graft a culture time of between 10 and 40 hours is suitable. The strain can, if desired, be concentrated by centrifugation or filtration followed by washing the concentrate in order to remove the culture medium. The concentration can then be frozen or lyophilized in the common way. In this way preparations of between 100 millions and 100,000 millions of living bacteria LMG-P-17806 per g can be prepared. A preparation can then be used as such or be used as an additive to food, for instance to milk or another product which gives LMG P-17806 the possibility to survive and, if desired, to grow.
Investigations
A. Investigation Concerning the Passage of the Strain Through the Gastrointestinal Tract
LMG- P-17806 was cultivated in the way described above and added together with yogurt culture to milk. A fermented product was produced by incubating the milk for five hours at +42xc2x0 C. A palatable product was obtained which contained fully 100 millions living LMG P-17806 per gram of product. Healthy persons were given 3xc3x97200 g product daily for one week. The total intake of LMG P-17806 was between 40 billions and 200 billions.
Faeces samples were examined before the intake, after one week of consumption and one week after the intake had ceased. As is evident from Tables 1 and 2 below, a strong increase in the number of lactobacilli in the test subjects was obtained. Two isolates per test subject were classified as to species on each occasion, i.e. 20 isolates in total. 18 of the isolates for the consumption time appeared to consist of LMG P-17806 according to fenotypical classification. This bacteria strain was not discovered in the samples before or after the intake of LMG P-17806.
In average the contents of the faeces samples during the supply were very high and varied only moderately from 63 millions to 320 millions per gram, i.e. with a factor of 5. Noteworthy was, that the contents were largely the same independent of what contents were measured before the start of the experiment. After the supply had ceased the contents reverted to what seems to be natural for the test subject in question.
Tables 1 and 2 below the content of lactobacilli in faeces determined, in millions per gram, by plating and using the substrate Rogosa.
The examination shows that the strain has good survival at the passage through the gastrointestinal tract.
B. Examination Concerning the Formation of L-lactic Acid by the Strain in a Model of Large Intestine
The fermented product above was added to a so called SHIME-reactor which is an in vitro model of the intestine. Samples were taken from the part of the reactor which corresponds to the most important parts of the large intestine. Similar comparative tests were carried out with some other Lacto bacillus strains, i.a. closely related L. paracasei subsp. paracasei. As is evident from the following Table 3 below LMG P-17806 gave a strong increase in the production of L-lactic acid, which is the very lactic acid isomer which is generated by LMG P-17806. A production of lactic acid is considered as favourable for several reasons, i.a. considering the anti-bacterial effect of the lactic acid as well as the fact that a lower pH is supposed to reduce the availability and formation of nitrogen compounds.
In the table xe2x80x9cLxe2x80x9d refers to the laevorotatory isomer of lactic acid and xe2x80x9cDxe2x80x9d to the dextrorotatory isomer.
Reactor 4 corresponds to the upper part of the large intestine, reactor 5 to the middle part and reactor 6 to the lower part of the large intestine. The investigation shows that the strain is forming L-lactic acid in the model of the large intestine.
C. Investigation of How the Strain is Protecting Intestinal Epithelium Cells from Invasion of Salmonella Typhimurium 
Intestinal epithelium cells of the type CaCo-2 cells were cultivated in-vitro. These were added with a combination of lactobacilli and Salmonella typhimurium in the ratio 100:1 with the addition of 1 million salmonella per ml. The effect was studied after incubation for 120 min at 370xc2x0 C. The amount of invading salmonella was determined by washing the plates with adhering CaCo-2 cells three times. The adhering cells were treated with the antibioticum gentamycin in a concentration of 100 mg/l for one hour in order to kill all bacteria which had not invaded cells. Then the plates were washed with PBS in order to remove all gentamycin and finally the entrapped bacteria were released by treating the CaCo-2 cells with 0.1% Triton-X during shaking. The number of salmonella was then determined by common plating methodology. LMG P-17806 had a pronounced effect in that it reduced a number of invaded cells. The closely related paracasei-variant 506 on the other hand seemed rather to stimulate the invasion of salmonella bacteria. Also with regard to this property LMG P-17806 showed a positive effect. The results are reported in Table 4 below.
The table shows that the strain LMG P-17806 gives a marked protection against invasion of salmonella bacteria.
D. Investigation of Protection Against Helicobacter Pylori 
In a mouse model where the mice had been infected with Helicobacter pylori the effect of supplying a fermented milk product with a strain of LMG P-17806 on one hand and without said strain on the other on the content of H. pylori measured in faeces was examined.
The mice were infected with 100 millions of the strain H. Pylori 17874 in helical form at three occasions with an interval of one day. Then the mice were given experimental products and the content of H. pylori in faeces was measured by means of heparinised magnetic balls and Enzyme Immuno Assay. Three products were examined. All the fermented milk products appeared to reduce the share of H. pylori, but the effect was occurring considerably faster in the cases when the product contained LMG P-17806 in comparison to common yoghurt and in comparison with a strain of L. fermentum KLD, respectively.
The figures in the table state the absorbency of 405 nm and are relative contents.
E. Examination of the Influence of the Strain LMG P-17806 on the Immunological Defence
The immunological defence system is controlled by a series of signal substances, so called cytokins. Some of these can be proinflammatory. The influence of LMG P-17806 on the production of cytokins TNF-alfa and IL-6 was compared with the influence of the two species contained in a yoghurt culture, L. delbruckei subsp. bulgaricus and Streptococcus thermophilus. Leucocytes were separated from human blood and added to living bacteria or bacteria killed with glutaraldehyde in an amount of 10 millions of leucocytes. As control lipopolysacharides (LPS) from E. coli were used. The results are reported in Table 6 below. The results show that the LMG P-17806 has the same inflammatory properties as a common yoghurt culture in the model used.
F. Examination of the Resistance of the Strain LMG P-17806 Against Antibiotics
Probiotics can be useful for use in connection with disorders in the balance of the intestine flora during medication with antibiotics. At the same time it is important that probiotics do not contribute to spreading of resistance to antibiotics, and this is especially important in the contemporary use of probiotics and antibiotics. The resistance of the strain LMG P-17806 against different antibiotics has been established for that reason. The sensitivity of the strain LMG P-17806 to different antibiotics were determined by establishing the content at which a reduction in the growth of the strain by 50%, measured as optical density, was obtained.
It appeared that the strain LMG P-17806 was resistant against vancomycin and was not inhibited even by 256 mg/l. The strain showed some resistance against trimethoprim and cefotaxime, an optical density (OD) of 50% at 12 and 4 mg/l, respectively being obtained. The strain LMG P-17806 was on the other hand sensitive to chloraphenicol, erythromcyin, rifampicin and tetracycline, where already levels below 1 mg/l resulted in an inhibition of the growth by 50%.
The influence by antibiotics was also examined as a survey by using so called Sensi discs from Oxoid. According to these results LMG P-17806 was resistant against aztreonam, ceftaximid, cefoxitin, colistine sulphate, kanamycin, polymyxin B, streptomycin and vancomycin.
LMG P-17806 might thus according to these results be especially interesting for use at the same time as therapy with antibiotics such as vancomycin, trimethoprim as well as several cefloxacins, which all are antibiotics with known side effects on the intestine flora and intestine function.
The Use of the Strain LMG P-17806